OBJECTIVETo study whether the analgesis of oxymatrine (OMT) affects N-type voltage-gated calcium channels (VGCCs).

METHODSTotally 45 mice were randomly divided into the sham-operation group, the model group [established by partial sciatic nerve ligation (PSNL)] , and the OMT treatment group according to random digit table, 15 in each group. The dorsal root ganglions (DRG) were separated in PSNL pain model mice. Intracellular calcium concentration ([Ca2+]i) was determined with Fluo-3 AM immunofluorescent probe in cultured DRG neurons. Different protein expression levels of N-type (Cav2. 2) and L-type ( Cav1. 3) among VGCCs from brain and DRG tissues were detected with Western blot.

RESULTSCompared with the sham-operation group, [Ca2+]i, increased in cultured DRG neurons (P <0. 05) , protein expression levels of Cav2. 2 in the brain tissue increased (P <0. 05), protein expression levels of Cav2. 2 in DRG tissues decreased in the model group (P <0. 01). Compared with the model group, [Ca2+]i, decreased in cultured DRG neurons (P < 0. 05), protein expression levels of Cav2. 2 in the brain tissue decreased (P <0. 01), protein expression levels of Cav2. 2 in DRG tissues increased in the OMT treatment group (P <0. 01). There was no statistical difference in Cav1. 3 expressions in cultured DRG neurons and the brain (P >0. 05).

CONCLUSIONAnalgesic effect of OMT might be related to Cav2. 2 channel mediated calcium ion flux.

Alkaloids ; pharmacology ; Analgesia ; methods ; Analgesics ; pharmacology ; Aniline Compounds ; Animals ; Calcium ; Calcium Channels, N-Type ; physiology ; Ganglia, Spinal ; Mice ; Neurons ; Pain ; Quinolizines ; pharmacology ; Xanthenes

Under some pathological conditions as bile flow obstruction or liver diseases with the enterohepatic circulation being disrupted, regurgitation of bile acids into the systemic circulation occurs and the plasma level of bile acids increases. Bile acids in circulation may affect the nervous system. We examined this possibility by studying the effects of bile acids on gating of neuronal (N)-type Ca2+ channel that is essential for neurotransmitter release at synapses of the peripheral and central nervous system. N-type Ca2+ channel currents were recorded from bullfrog sympathetic neuron under a cell-attached mode using 100 mM Ba2+ as a charge carrier. Cholic acid (CA, 10(-6) M) that is relatively hydrophilic thus less cytotoxic was included in the pipette solution. CA suppressed the open probability of N-type Ca2+ channel, which appeared to be due to an increase in null (no activity) sweeps. For example, the proportion of null sweep in the presence of CA was ~40% at +40 mV as compared with ~8% in the control recorded without CA. Other single channel properties including slope conductance, single channel current amplitude, open and shut times were not significantly affected by CA being present. The results suggest that CA could modulate N-type Ca2+ channel gating at a concentration as low as 10(-6) M. Bile acids have been shown to activate nonselective cation conductance and depolarize the cell membrane. Under pathological conditions with increased circulating bile acids, CA suppression of N-type Ca2+ channel function may be beneficial against overexcitation of the synapses.
Bile ; Bile Acids and Salts ; Calcium Channels, N-Type ; Cell Membrane ; Central Nervous System ; Cholic Acid ; Enterohepatic Circulation ; Fees and Charges ; Ganglia, Sympathetic ; Liver Diseases ; Nervous System ; Neurons ; Neurotransmitter Agents ; Plasma ; Rana catesbeiana ; Synapses

Voltage dependent calcium channel (VDCC), one of the most important regulator of Ca2+ concentration in neuron, play an essential role in the central processing of nociceptive information. The present study investigated the antinociceptive effects of L, T or N type VDCC blockers on the formalin-induced orofacial inflammatory pain. Experiments were carried out on adult male Sprague-Dawley rats weighing 220-280 g. Anesthetized rats were individually fixed on a stereotaxic frame and a polyethylene (PE) tube was implanted for intracisternal injection. After 72 hours, 5% formalin (50 microL) was applied subcutaneously to the vibrissa pad and nociceptive scratching behavior was recorded for nine successive 5 min intervals. VDCC blockers were administered intracisternally 20 minutes prior to subcutaneous injection of formalin into the orofacial area. The intracisternal administration of 350 or 700 microg of verapamil, a blocker of L type VDCC, significantly decreased the number of scratches and duration in the behavioral responses produced by formalin injection. Intracisternal administration of 75 or 150 microg of mibefradil, a T type VDCC blocker, or 11 or 22 microg of cilnidipine, a N type VDCC blocker, also produced significant suppression of the number of scratches and duration of scratching in the first and second phase. Neither intracisternal administration of all VDCC blockers nor vehicle did not affect in motor dysfunction. The present results suggest that central VDCCs play an important role in orofacial nociceptive transmission and a targeted inhibition of the VDCCs is a potentially important treatment approach for inflammatory pain originating in the orofacial area.
Adult ; Animals ; Calcium ; Calcium Channel Blockers ; Calcium Channels ; Calcium Channels, L-Type ; Calcium Channels, N-Type ; Calcium Channels, T-Type ; Dihydropyridines ; Facial Pain ; Formaldehyde ; Humans ; Injections, Subcutaneous ; Male ; Mibefradil ; Neurons ; Pain Measurement ; Polyethylene ; Rats ; Rats, Sprague-Dawley ; Verapamil

OBJECTIVETo compare the effects of amlodipine, benidipine and nifedipine on myocardial hypertrophy and evaluate the underlying mechanism.

METHODSMyocardial hypertrophy model was created by transverse aortic constriction (TAC) in C57 BL/6 mice, and plasma catecholamine concentrations were measured 7 days after surgery to confirm the sympathetic activation. The 3 drugs were administered in TAC mice for 7 days and cardiac hypertrophy was evaluated according to the heart-to-body weight ratio (HW/BW). Effects of those drugs on the protein synthesis stimulated by phenylephrine in cultured neonatal cardiac myocytes were also examined.

RESULTSHW/BW and plasma concentrations of catecholamine were significantly increased in TAC mice one week after surgery in comparison with to sham-operated mice. One week after TAC, the HW/BW ratio was significantly lower in the amolodipine but not nifedipine-treated group than in the TAC group. Administration of nifedipine via minipump infusion for one week did not decrease HW/BW ratio. Treatment with amlodpine or benidipine, but not nifedipine, decreased the neonatal rat myocyte protein synthesis induced by phenylephrine stimulation.

CONCLUSIONAntihypertrophic effect of DHEs on myocardium is dependent on their potential of blocking N-type calcium channel, and the underlying mechanism involves the sympathetic inhibition.

Amlodipine ; pharmacology ; therapeutic use ; Animals ; Calcium Channel Blockers ; pharmacology ; therapeutic use ; Calcium Channels, N-Type ; drug effects ; Cardiomegaly ; drug therapy ; etiology ; Dihydropyridines ; pharmacology ; therapeutic use ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred C57BL ; Nifedipine ; pharmacology ; therapeutic use

OBJECTIVE: To prove the existence neurons in the rat corpus callosum, the potential function of these neurons and their connection. METHODS: Immunohistochemistry was used performed to examine the expressions of NeuN, a mature neuron marker,and N-type voltage-dependent valcium channel alpha1-subunit (Cav2.2)in the section of the rat corpus callosum. Horseradish peroxidase (HRP) normal sodium solution (30%), the retrograde tracer,was injected under the frontal forceps of corpus callousm and HRP absorbed by the process of neurons in the brain slices was stained with tetramethyl benzidine. RESULTS: There were some NeuN positive cells in the rat corpus callosum and Cav2.2 was detected in some of these NeuN positive cells.Neurons with positive HRP were found in the rat corpus callosum and some of these neurons connected to the cortex or corpus striatum. CONCLUSION There are a few neurons in the corpus callosum of adult rats and some of them express Cav2.2. Neurons in the corpus callosum have connections with the brain cortex or corpora striatum.
Animals ; Calcium Channels, N-Type ; biosynthesis ; Corpus Callosum ; cytology ; metabolism ; DNA-Binding Proteins ; Male ; Nerve Tissue Proteins ; biosynthesis ; Neural Pathways ; physiology ; Neurons ; cytology ; Nuclear Proteins ; biosynthesis ; Random Allocation ; Rats ; Rats, Sprague-Dawley

The present study was designed to establish comparatively the inhibitory effects of cilnidipine (CNP), nifedipine (NIF), and omega-conotoxin GVIA (CTX) on the release of CA evoked by cholinergic stimulation and membrane depolarization from the isolated perfused model of the rat adrenal medulla. CNP (3 micrometer), NIF (3 micrometer), and CTX (3 micrometer) perfused into an adrenal vein for 60 min produced greatly inhibition in CA secretory responses evoked by ACh (5.32 x 10(-3) M), DMPP (10(-4) M for 2 min), McN-A-343 (10(-4) M for 2 min), high K+ (5.6 x 10(-2) M), Bay-K-8644 (10(-5) M), and cyclopiazonic acid (10(-5) M), respectively. For the CA release evoked by ACh and Bay-K-8644, the following rank order of potency was obtained: CNP > NIF > CTX. The rank order for the CA release evoked by McN-A-343 and cyclopiazonic acid was CNP > NIF > CTX. Also, the rank orders for high K+ and for DMPP were NIF > CTX > CNP and NIF > CNP > CTX, respectively. Taken together, these results demonstrate that all voltage-dependent Ca2+ channels (VDCCs) blockers of cilnidipine, nifedipine, and omega-conotoxin GVIA inhibit greatly the CA release evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors and the membrane depolarization without affecting the basal release from the isolated perfused rat adrenal gland. It seems likely that the inhibitory effects of cilnidipine, nifedipine, and omega-conotoxin GVIA are mediated by the blockade of both L- and N-type, L-type only, and N-type only VDCCs located on the rat adrenomedullary chromaffin cells, respectively, which are relevant to Ca2+ mobilization. It is also suggested that N-type VDCCs play an important role in the rat adrenomedullary CA secretion, in addition to L-type VDCCs.
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride ; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; Adrenal Glands ; Adrenal Medulla* ; Animals ; Calcium Channels ; Calcium Channels, L-Type ; Calcium Channels, N-Type ; Chromaffin Cells ; Dimethylphenylpiperazinium Iodide ; Membranes ; Nifedipine* ; omega-Conotoxin GVIA* ; omega-Conotoxins* ; Rats* ; Veins

Melittin-induced nociceptive responses are mediated by selective activation of capsaicin-sensitive primary afferent fibers and are modulated by excitatory amino acid receptor, cyclooxygenase, protein kinase C and serotonin receptor. The present study was undertaken to investigate the peripheral and spinal actions of voltage-gated calcium channel antagonists on melittin-induced nociceptive responses. Changes in mechanical threshold and number of flinchings were measured after intraplantar (i.pl.) injection of melittin (30microg/paw) into mid-plantar area of hindpaw. L-type calcium channel antagonists, verapamil [intrathecal (i.t.), 6 or 12microg; i.pl.,100 & 200microg; i.p., 10 or 30 mg], N-type calcium channel blocker, omega-conotoxin GVIA (i.t., 0.1 or 0.5microg; i.pl., 5microg) and P-type calcium channel antagonist, omega-agatoxin IVA (i.t., 0.5microg; i.pl., 5microg) were administered 20 min before or 60 min after i.pl. injection of melittin. Intraplantar pre-treatment and i.t. pre- or post-treatment of verapamil and omega-conotoxin GVIA dose-dependently attenuated the reduction of mechanical threshold, and melittin-induced flinchings were inhibited by i.pl. or i.t. pre-treatment of both antagonists. P-type calcium channel blocker, omega-agatoxin IVA, had significant inhibitory action on flinching behaviors, but had a limited effect on melittin-induced decrease in mechanical threshold. These experimental findings suggest that verapamil and omega-conotoxin GVIA can inhibit the development and maintenance of melittin-induced nociceptive responses.
Animals ; Calcium Channels* ; Calcium Channels, L-Type ; Calcium Channels, N-Type ; Calcium Channels, P-Type ; Calcium* ; Hyperalgesia ; Ions* ; Melitten ; Nociception* ; omega-Agatoxin IVA ; omega-Conotoxin GVIA ; Prostaglandin-Endoperoxide Synthases ; Protein Kinase C ; Rats* ; Receptors, Glutamate ; Serotonin ; Verapamil

AIM AND METHODSWhole-cell recording technique was used to observe the changes of voltage-dependent ion channels and NMDA receptor currents of rat hippocampal neurons during primary culture.

RESULTSThere was no significant difference of voltage-dependent Na+ current (I(Na)) at 7 d, 14 d and 21 d in culture. It's the same for delayed rectifier K+ current (Ik). However, voltage-dependent Ca2+ current (I(Ca)) and its density were continuously and markedly increased. Further studies showed that the increase of I(Ca) was resulted from the increase of L-type voltage-dependent Ca2+ channels (L-VDCC). NMDA receptor current was also significantly increased with time of culture.

CONCLUSIONCa2+ influx through VDCC and NMIDA receptor is the fatal factor in the aging and death of hippocampal neurons.

Animals ; Animals, Newborn ; Calcium ; metabolism ; Calcium Channels, L-Type ; metabolism ; Cell Membrane ; metabolism ; Cells, Cultured ; Cellular Senescence ; Hippocampus ; cytology ; Ion Channels ; metabolism ; Neurons ; metabolism ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Time Factors

To evaluate the effects of different antagonists on the release of cytochrome c from mitochondria to cytosol and the expression of Bcl-2 in mitochondria in rat hippocampus after ischemia, we examined Bcl-2 and cytochrome c expression by immunoblotting using 4-vessel occlusion (4-VO) as brain ischemia model. The results showed that after 24 h ischemia/reperfusion (I/R) cytochrome c decreased markedly in mitochondria, which was correspondingly increased in the cytosolic fraction. Bcl-2 expression was time-dependent, reaching its peak level after 6 h I/R. In all those samples, there were no alterations in the subcellular distribution of cytochrome oxidase, a mitochondrial respiratory chain protein. The decreases in Bcl-2 and cytochrome c in mitochondria were restored by pretreatment with non-competitive NMDA receptor antagonist ketamine or L-type voltage-gated Ca(2+) channel (L-VGCC) antagonist nifedipine at 20 min prior to ischemia. The results demonstrate that the release of cytochrome c from mitochondria to cytosol and the up-regulation of Bcl-2 are possibly mediated by NMDA receptors or L-VGCC following brain ischemia. Cytochrome c release may be injurious while Bcl-2 up-regulation may be protective to ischemic hippocampus.
Animals ; Brain Ischemia ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; drug effects ; Cytochromes c ; metabolism ; Cytosol ; Hippocampus ; metabolism ; Ketamine ; pharmacology ; Male ; Mitochondria ; metabolism ; Nifedipine ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; Up-Regulation

The present study was attempted to investigate the effect of cilnidipine (FRC-8635), which is a newly synthesized novel dihydropyridine (DHP) type of organic Ca2 channel blockers, on secretion of catecholamines (CA) evoked by acetylcholine (ACh), high K, DMPP and McN-A-343 from the isolated perfused rat adrenal gland. Cilnidipine (1~10microM) perfused into an adrenal vein for 60 min produced relatively dose- and time-dependent inhibition in CA secretory responses evoked by ACh (5.32 10 3 M), DMPP (10 4 M for 2 min) and McN-A-343 (10 4 M for 2 min). However, lower dose of cilnidipine did not affect CA secretion by high K (5.6 10 2 M), higher dose of it reduced greatly CA secretion of high K. Cilnidipine itself did fail to affect basal catecholamine output. In the presence of cilnidipine (10microM), the CA secretory responses evoked by Bay-K-8644 (10microM), an activator of L-type Ca2 channels and cyclopiazonic acid (10microM), an inhibitor of cytoplasmic Ca2 -ATPase were also inhibited. Moreover, omega-conotoxin GVIA (1microM), a selective blocker of the N-type Ca2 channels, given into the adrenal gland for 60 min, also inhibited time-dependently CA secretory responses evoked by Ach, high K, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid. Taken together, these results demostrate that cilnidipine inhibits CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors from the isolated perfused rat adrenal gland without affecting the basal release. However, at lower dose, cilnidipine did not affect CA release by membrane depolarization while at larger dose inhibited that. It seems likely that this inhibitory effect of cilnidipine is exerted by blocking both L- and N-type voltage-dependent Ca2 channels (VDCCs) on the rat adrenomedullary chromaffin cells, which is relevant to inhibition of both the Ca2 influx into the adrenal chromaffin cells and intracellular Ca2 release from the cytoplasmic store. It is thought that N-type VDCCs may play an important role in regulation of CA release from the rat adrenal medulla.
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride ; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; Acetylcholine ; Adrenal Glands ; Adrenal Medulla* ; Animals ; Calcium Channels, N-Type ; Catecholamines ; Chromaffin Cells ; Cytoplasm ; Dimethylphenylpiperazinium Iodide ; Membranes ; omega-Conotoxin GVIA ; Rats* ; Veins